Selective protein degradation plays a direct role in regulating many cellular processes, including cell-cycle progression, normal and aberrant growth and differentiation, the stress response, and the elimination of damaged or otherwise abnormal proteins. However, little is known about the mechanisms governing the substrate selectivity of proteolytic pathways in eucaryotes. The objective of the research proposed here is to explore in mammalian cells the regulation and mechanism of substrate selection by the N-end rule pathway, a ubiquitin-dependent proteolytic pathway whose substrate selectivity is governed by the identity of the substrate protein's amino-terminal residue. Summary of specific aims: (1) To determine the selectivity of the N-end rule pathway in proliferating and differentiating murine erythroleukemia cells, to directly test the conjecture (Gonda et al., 1989) that the selectivity of ubiquitin-dependent protein degradation in erythroid cells is differentiation specific. (2) To clone cDNAs encoding the E3alpha protein, one of the ubiquitin- protein ligases which governs substrate selection in the N-end rule pathway; (3) To overexpress and purify the E3alpha protein for biochemical analyses; (4) To functionally dissect the E3alpha protein, using both limited proteolysis and expression of mutant gene products; (5) To identify genes encoding other E3 proteins that govern substrate selection in the ubiquitin system, via homology with the cDNAs encoding E3alpha The proposed work will test specific hypotheses regarding the role of ubiquitin-dependent proteolysis and the N-end rule in erythroid differentiation, and will initiate the molecular analysis of substrate selection by the ubiquitin system in mammalian cells. Understanding the regulation of selective proteolysis is needed to understand the regulation of many normal and abnormal aspects of cellular physiology, including aging, carcinogenesis, growth and differentiation.